Fluorescence reflectance imaging of macrophage-rich atherosclerotic plaques using an alphavbeta3 integrin-targeted fluorochrome.
Macrophages play an important role during the development and progression of atherosclerotic plaques. alphavbeta3 integrins are highly expressed by macrophages; thus, targeting alphavbeta3 may allow targeting of culprit macrophage-loaded atherosclerotic lesions in vivo.


METHODS
An alphavbeta3-targeted Arg-Gly-Asp (RGD) peptide was labeled with the cyanine 5.5 (Cy 5.5) dye and applied to image atherosclerotic plaques in apolipoprotein E-deficient mice.


RESULTS
The peptide-dye conjugate binds to alphavbeta3 integrin-positive RAW264.7 macrophages with high affinity. Competition experiments confirmed binding specificity of the probe. A significant fluorochrome accumulation in atherosclerotic plaques was demonstrated 24 h after injection by fluorescence reflectance imaging, which was blocked with high efficiency by competition with the unlabeled peptide. Conversely, the nonconjugated dye revealed only a minor fluorescence signal in the plaques. Fluorescence microscopy revealed colocalization of the probe with macrophages in the plaque of a mouse model for accelerated atherosclerosis, which was corroborated in human carotid artery specimens. In addition to macrophage-associated signals, binding of the probe to the neointima or elastica of the arteries was observed.


CONCLUSIONS
RGD-Cy 5.5, combined with near-infrared optical imaging methods, allows the specific imaging of alphavbeta3-integrin expression on macrophages recruited to vascular lesions and may serve to estimate macrophage-bound inflammatory activity of atherosclerotic lesions.
Waldeck
Jens
J
Department of Clinical Radiology, University Hospital Muenster, University of Muenster, Muenster, Germany. waldeck@uni-muenster.de
Häger
Florian
F
Höltke
Carsten
C
Lanckohr
Christian
C
von Wallbrunn
Angelika
A
Torsello
Giovanni
G
Heindel
Walter
W
Theilmeier
Gregor
G
Schäfers
Michael
M
Bremer
Christoph
C
D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency; D050197:Atherosclerosis / Q000378:metabolism* / Q000473:pathology*; D002232:Carbocyanines / Q000378:metabolism; D002460:Cell Line; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007259:Infrared Rays; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D010456:Peptides, Cyclic / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D001057:Apolipoproteins E; D002232:Carbocyanines; D005456:Fluorescent Dyes; D039302:Integrin alphaVbeta3; D010456:Peptides, Cyclic; C085321:cyanine dye 5; C507169:cyclic arginine-glycine-aspartic acid peptide